Imunogenic conjugate of carbohydrate haptens and aggregated protein carrier

Abstract

A conjugate of a plurality of carbohydrate haptens and an aggregate (multimer) of monomeric units of a carrier moiety is provided. The conjugate may be used to elicit an immune response. The conjugate is preferably a carbohydrate-substituted aggregate of KLH monomers, in particular, a dimeric, trimeric or tetrameric aggregated. An aggregated STn-KLH conjugate is of particular interest.

1 . A non-naturally occurring immunogenic conjugate comprising a plurality carbohydrate hapten moieties, which may be the same or different, and of a plurality of monomeric units of a lysine-containing immunogenic protein carrier moiety, said hapten moieties being attached, directly or indirectly, to lysines of the monomeric units of the carrier moiety.  
     
     
         2 . The conjugate of  claim 1  where said carrier moiety is keyhole limpet hemocyanin.  
     
     
         3 . The conjugate of  claim 1  which has an apparent molecular weight more than 800 kD.  
     
     
         4 . The conjugate of  claim 1  which has an apparent molecular weight of more than 1,200 kD.  
     
     
         5 . The conjugate of  claim 1  which has an apparent molecular weight of more than 1,600 kD.  
     
     
         6 . The conjugate of  claim 1  which has an apparent molecular weight of at least 1,000 kD.  
     
     
         7 . The conjugate of  claim 1  which has an apparent molecular weight of at least 1,500 kD.  
     
     
         8 . The conjugate of  claim 1  which has an apparent molecular weight of at least 2,000 kD.  
     
     
         9 . The conjugate of  claim 1  which has an apparent molecular weight of not more than 5,000 kD.  
     
     
         10 . The conjugate of  claim 1  which has an apparent molecular weight of not more than 2,500 kD.  
     
     
         11 . The conjugate of  claim 1  whose immunogenic potency is at least 200% that of a reference conjugate consisting of said hapten moieties and a single monomeric unit of said carrier moiety with the same expected number of hapten moieties per monomeric unit of carrier.  
     
     
         12 . The conjugate of  claim 1  where the expected number of hapten moieties per monomeric unit of carrier is at least 10.  
     
     
         13 . The conjugate of  claim 1  where one or more said carbohydrate hapten moieties provides a sialyl Tn epitope.  
     
     
         14 . The conjugate of  claim 13  where the NANA content is in excess of 3%, measured as amount of sialic acid as percentage of the protein content of the conjugate (weight/weight).  
     
     
         15 . The conjugate of  claim 13  where the NANA content is at least 4%.  
     
     
         16 . The conjugate of  claim 13  where the NANA content is at least 5%.  
     
     
         17 . The conjugate of  claim 13  where the NANA content is at least 6%.  
     
     
         18 . The conjugate of  claim 13  where the NANA content is at least 7%.  
     
     
         19 . The conjugate of  claim 1  where each carbohydrate hapten moiety is attached to a monomeric unit of the carrier moiety by a linker.  
     
     
         20 . The conjugate of  claim 19  where the linker is an alphatic group consisting of not more than 12 atoms other than hydrogen.  
     
     
         21 . The conjugate of  claim 20  where the linker is an alkyl group.  
     
     
         22 . The conjugate of  claim 19  where the linker connects an oxygen of the hapten moiety to an amino nitrogen.  
     
     
         23 . The conjugate of  claim 19  where a linker connects a single hapten moiety to a single attachment site on said monomeric unit.  
     
     
         24 . The conjugate of  claim 19  where a linker connects a plurality of hapten moieties to a single attachment site on said monomeric unit.  
     
     
         25 . The conjugate of  claim 1  where at least one of said carbohydrate hapten moieties is not natively associated with KLH.  
     
     
         26 . A composition comprising conjugate molecules of  claim 1  wherein said conjugate molecules are at least 50% by weight of all conjugate molecules in said composition which comprise said carbohydrate hapten moieties.  
     
     
         27 . A composition comprising conjugate molecules according to  claim 1  where said conjugate molecules are at least 75% by weight of all conjugates in said composition which comprise said carbohydrate hapten moieties.  
     
     
         28 . A composition comprising conjugate molecules according to claim  1  where said conjugate molecules are at least 90% by weight of all conjugate molecules in said composition which comprise said carbohydrate hapten moieties.  
     
     
         29 . A composition comprising conjugate molecules according to  claim 1  where said conjugate molecules are at least 95% by weight of all conjugate molecules in said composition which comprise said carbohydrate hapten moieties.  
     
     
         30 . A method of preparing the conjugate of  claim 1  which comprises a conjugation step which comprises reacting one or more reagents, each comprising at least one of said carbohydrate hapten moieties, with said monomeric units, or with a preaggregated carrier moiety comprising a plurality of said units, so as to obtain said conjugate.  
     
     
         31 . The method of  claim 30  where said reagent is the aldehyde derivative of said carbohydrate hapten.  
     
     
         32 . The method of  claim 31  where said aldehyde derivative is obtained by provide a crotyl derivative of the hapten, ozonoyzing the crotyl derivative to obtain an ozonide, and reducing the ozonide to form the aldehyde derivative.  
     
     
         33 . The method of  claim 32  in which acetaldehyde is provided as a byproduct to production of the hapten aldehyde.  
     
     
         34 . The method of  claim 30  where, in the conjugation step, the reaction temperature is 39° C. to 45° C. and the reaction time is 17 hours to 25 hours.  
     
     
         35 . The method of  claim 30  where, in the conjugation step, the reaction temperature is greater than 26° C.  
     
     
         36 . The method of  claim 30  where, in the conjugation step, the reaction temperature is at least 30° C.  
     
     
         37 . The method of  claim 30  where, in the conjugation step, the reaction temperature is at least 35° C.  
     
     
         38 . The method of  claim 30  where, in the conjugation step, the reaction temperature is at least 39° C.  
     
     
         39 . The method of  claim 30  where, in the conjugation step, the reaction temperature is not greater than 45° C.  
     
     
         40 . The method of  claim 30  where, in the conjugation step, the reaction temperature is not greater than 43° C.  
     
     
         41 . The method of  claim 30  where, in the conjugation step, the reaction time is more than 6 hours.  
     
     
         42 . The method of  claim 30  where, in the conjugation step, the reaction time is at least 12 hours.  
     
     
         43 . The method of  claim 30  where, in the conjugation step, the reaction time is at least 17 hours.  
     
     
         44 . The method of  claim 30  where, in the conjugation step, the reaction time is not more than 40 hours.  
     
     
         45 . The method of  claim 30  where, in the conjugation step, the reaction time is not more than 30 hours.  
     
     
         46 . The method of  claim 30  where, in the conjugation step, the reaction time is not more than 25 hours.  
     
     
         47 . The method of  claim 30  where the weight ratio of the hapten-aldehyde to the carrier monomer is at least 0.6:1.  
     
     
         48 . The method of  claim 30  where the weight ratio of the hapten-aldehyde to the carrier monomer is at least 1:1.  
     
     
         49 . The method of  claim 30  where the weight ratio of the hapten-aldehyde to the carrier monomer is at least 1.5:1.  
     
     
         50 . The method of  claim 30  where the weight ratio of the hapten-aldehyde to the carrier monomer is at least 1.75:1.  
     
     
         51 . The method of  claim 30  where the weight ratio of the hapten-aldehyde to the carrier monomer is at least 2:1.  
     
     
         52 . The method of  claim 30  where the weight ratio of the hapten-aldehyde to the carrier monomer is not more than 4:1.  
     
     
         53 . The method of  claim 30  where the weight ratio of the hapten-aldehyde to the carrier monomer is at least 3.25:1.  
     
     
         54 . The method of  claim 30  where the weight ratio of the hapten-aldehyde to the carrier monomer is at least 2.75:1.  
     
     
         55 . The method of  claim 30  where the weight ratio of the hapten-aldehyde to the carrier monomer is about 2.25:1.  
     
     
         56 . The method of  claim 30  where said conjugation step comprises production of a schiff base intermediate, and the intermediate is reduced with a reducing agent.  
     
     
         57 . The method of  claim 56  in which the reducing agent is sodium cyanoborohydride.  
     
     
         58 . The method of  claim 57  in which the reducing agent is used in a final concentration of 25-125 mM.  
     
     
         59 . The method of  claim 57  in which the reducing agent is used in a final concentration of 45-75 mM.  
     
     
         60 . A method of eliciting a carbohydrate hapten-specific immune response in a subject which comprises administering to the subject an immunologically effective amount of the conjugate of  claim 1 .  
     
     
         61 . The method of  claim 60  in which a humoral immune response is elicited.  
     
     
         62 . The method of  claim 60  in which a cellular immune response is elicited.  
     
     
         63 . The method of  claim 60  in which the carbohydrate hapten is tumor-associated.  
     
     
         64 . The method of  claim 60  where said conjugate or composition is protective against a disease.  
     
     
         65 . The method of  claim 64  in which the disease is a cancer.  
     
     
         66 - 67  (cancelled)  
     
     
         68 . The conjugate of  claim 1  where the conjugate comprises at least three monomer units of the carrier moiety.  
     
     
         69 . The conjugate of  claim 1  where the conjugate comprises at least four monomer units of the carrier moiety.  
     
     
         70 . The composition of  claim 26  where the conjugate comprises at least three monomer units of the carrier moiety.  
     
     
         71 . The composition of  claim 26  where the conjugate comprises at least four monomer units of the carrier moiety.  
     
     
         72 . The conjugate of  claim 1  wherein the monomeric unit of the carrier moiety is the monomeric unit of a hemocyanin.  
     
     
         73 . The conjugate of  claim 72  wherein the hemocyanin is an arthropod hemocyanin.  
     
     
         74 . The conjugate of  claim 72  where the hemocyanin is a molluscan hemocyanin.  
     
     
         75 . The conjugate of  claim 72  where the hemocyanin is a gastropod hemocyanin.  
     
     
         76 . The conjugate of  claim 72  where the hemocyanin is  Fissurellidae  hemocyanin.  
     
     
         77 . The conjugate of  claim 2  where the carrier moiety of the conjugate is not a decamer, didecamer, or multidecamer of the monomeric unit of KLH.  
     
     
         78 . The conjugate of  claim 2  where the carrier moiety of the conjugate is not a dimer of the monomeric unit of KLH.  
     
     
         79 . The conjugate of  claim 2  where the carrier moiety of the conjugate is a dimer, trimer, tetramer or pentamer of the monomeric unit of KLH.  
     
     
         80 . The conjugate of  claim 1  wherein at least one carbohydrate hapten moiety comprise the TF disaccharide.  
     
     
         81 . The conjugate of  claim 1  wherein at least one carbohydrate hapten moiety comprises Tn.  
     
     
         82 . The conjugate of  claim 1  wherein at least one carbohydrate hapten moiety is a sialylated carbohydrate.